Long-term Incubation Research Articles

Asynchronous characteristics of Feammox and iron reduction from paddy soils in Southern China

Recently, the newly discovered anaerobic ammonium oxidation coupled with iron reduction (i.e., Feammox) has been proven to be a widespread nitrogen (N) loss pathway in ecosystems and has an essential contribution to gaseous N loss in paddy soil. However, the mechanism of iron-nitrogen coupling transformation and the role of iron-reducing bacteria (IRB) in Feammox were poorly understood. This study investigated the Feammox and iron reduction changes and microbial community evolution in a long-term anaerobic incubation by 15N isotope labeling combined with molecular biological techniques. The average rates of Feammox and iron reduction during the whole incubation were 0.25 ± 0.04 μg N g−1 d−1 and 40.58 ± 3.28 μg Fe g−1 d−1, respectively. High iron oxide content increased the Feammox rate, but decreased the proportion of Feammox–N2 in three Feammox pathways. RBG-13-54-9, Brevundimonas, and Pelomonas played a vital role in the evolution of microbial communities. The characteristics of asynchronous changes between Feammox and iron reduction were found through long-term incubation. IRB might not be the key species directly driving Feammox, and it is necessary to reevaluate the role of IRB in Feammox process.

Probiotic characterization of a novel Bacillus cereus strain fkW8-1-2 isolated from intestine of white crucian carp (Carassius cuvieri)

Microbial infection can pose a great threat to health of farmed fish. This study aimed to investigate the probiotic characteristics of Bacillus cereus strain fkW8-1-2 isolated from intestine of WCC and its anti-biofilm activity against pathogenic bacteria. The strain fkW8-1-2 showed a high resistance to low pH, bile salt and lysozyme during long-term incubation. The strain fkW8-1-2 displayed a gradual increase of cell surface hydrophobicity with various forms of hydrocarbons. The strain fkW8-1-2 showed the remarkable autoaggregation and coaggregation activity with A. hydrophila L3-3 and E. tarda 1l-4. The culture medium (CM) and cell-free supernatants (CFS) of strain fkW8-1-2 at 48 h cultivation could attenuate biofilm formation of A. hydrophila and E. tarda with the maximum inhibition rates. In addition, the intact cells of strain fkW8-1-2 exhibited the strong antioxidant activity for DPPH removal and superoxide anion clearance. These results indicated that strain fkW8-1-2 possessing probiotic characteristics can pose a protective role in health of farmed fish.

The Effects of the Steroids 5-Androstenediol and Dehydroepiandrosterone and Their Synthetic Derivatives on the Viability of K562, HeLa, and Wi-38 Cells and the Luminol-Stimulated Chemiluminescence of Peripheral Blood Mononuclear Cells from Healthy Volunteers.

In order to evaluate the role of substituents at 3-C and 17-C in the cytotoxic and cytoprotective actions of DHEA and 5-AED molecules, their derivatives were synthesized by esterification using the corresponding acid anhydrides or acid chlorides. As a result, seven compounds were obtained: four DHEA derivatives (DHEA 3-propionate, DHEA 3-butanoate, DHEA 3-acetate, DHEA 3-methylsulfonate) and three 5-AED derivatives (5-AED 3-butanoate, 5-AED 3,17-dipropionate, 5-AED 3,17-dibutanoate). All of these compounds showed micromolar cytotoxic activity toward HeLa and K562 human cancer cells. The maximum cytostatic effect during long-term incubation for five days with HeLa and K562 cells was demonstrated by the propionic esters of the steroids: DHEA 3-propionate and 5-AED 3,17-dipropionate. These compounds stimulated the growth of normal Wi-38 cells by 30-50%, which indicates their cytoprotective properties toward noncancerous cells. The synthesized steroid derivatives exhibited antioxidant activity by reducing the production of reactive oxygen species (ROS) by peripheral blood mononuclear cells from healthy volunteers, as demonstrated in a luminol-stimulated chemiluminescence assay. The highest antioxidant effects were shown for the propionate ester of the steroid DHEA. DHEA 3-propionate inhibited luminol-stimulated chemiluminescence by 73% compared to the control, DHEA, which inhibited it only by 15%. These data show the promise of propionic substituents at 3-C and 17-C in steroid molecules for the creation of immunostimulatory and cytoprotective substances with antioxidant properties.

Effects of biobased fertilisers on soil physical, chemical and biological indicators – a one-year incubation study

Soil quality is declining in Europe and globally due to agricultural practices and climate change. The European market for novel biobased fertilisers (BBFs) is growing and the new European Union fertiliser regulation promotes their use. However, knowledge about the effects of many novel BBFs on soil quality is currently very limited. In a one-year laboratory incubation experiment, this study aimed to test the effect on biological (microbial biomass carbon (C)), physical (clay dispersibility and water-holding capacity) and chemical (pH, cation exchange capacity (CEC), total C and C in soil size fractions (<250, 50–250 and >50 μm)) soil quality indicators of 10 BBFs applied at two different rates on two soil types: an Arenosol and a Luvisol. The set-up also included a soil that was subjected to long-term annual application of the compost used in the incubation. The application of BBFs generally improved soil quality, with the compost material improving soil quality most, followed by a plant-based fertiliser and a biogas digestate. The effect of BBF application on CEC, total C and particulate organic matter (POM) was related to the amount of total C added with the BBF. Furthermore, the effect on total C and POM fractions was also related to easily decomposable C added with the BBF. Comparing the single accelerated application with annual application under field conditions indicated that the long-term incubation trial is a reasonable predictor of compost long-term effects in the field. Whether this applies to BBFs with very different properties remains to be shown.

Influence and Mechanism Study of Soil Moisture on the Stability of Arsenic-Bearing Ferrihydrite in Surface Soil Vertical Profiles

Ferrihydrite is usually used as a remedy for arsenic (As)-contaminated soil due to its strong affinity and large specific surface area. However, its noncrystalline phase makes it unstable in long-term applications in the soil. In this study, a soil incubation experiment was designed using the diffusive gradient in thin film (DGT) technique and spectral techniques to investigate the fate of As-bearing ferrihydrite [As(V)-Fh] after long-term incubation at different soil water holding capacities (SWHCs). After As(V)-Fh (0.05 and 0.005 As/Fe molar ratio) was incubated in soil for 360 days, both DGT-derived labile As and Fe were released at 70% SWHC and 120% SWHC into the soil (at a vertical depth of 12 cm). The concentrations of DGT-As and DGT-Fe increased with incubation time and were greater at 120% SWHC. The results of X-ray diffraction (XRD) and scanning electron microscopy (SEM) showed that As(V)-Fh gradually transformed into hematite and goethite after 360 days of incubation. Goethite was mainly found in the 120% SWHC treatment after 180 days. Hematite and goethite formation rates were greater in the 120% SWHC treatment and in the bottom soil layer. Mechanistic analysis based on X-ray photoelectron spectroscopy (XPS) revealed that the variation in soil pH and the formation of Fe(II) (under flooded water conditions) are the two key factors promoting the formation of hematite (dehydrogenation and dehydration) and goethite (As(V)-Fh dissociation and reorganization). The As release mainly occurred due to the loss of adsorption sites. Thus, it is recommended that ferrihydrite be applied in paddy–dry rotations or dry-field patterns to effectively avoid the loss of As(V)-Fh in long-term-saturated soil.

Quantifying the δ15N trophic offset in a cold-water scleractinian coral (CWC): implications for the CWC diet and coral δ15N as a marine N cycle proxy

Abstract. The nitrogen (N) isotope composition (δ15N) of cold-water corals is a promising proxy for reconstructing past ocean N cycling, as a strong correlation was found between the δ15N of the organic nitrogen preserved in coral skeletons and the δ15N of particulate organic matter exported from the surface ocean. However, a large offset of 8 ‰–9 ‰ between the δ15N recorded by the coral and that of exported particulate organic matter remains unexplained. The 8 ‰–9 ‰ offset may signal a higher trophic level of coral dietary sources, an unusually large trophic isotope effect or a biosynthetic δ15N offset between the coral's soft tissue and skeletal organic matter, or some combinations of these factors. To understand the origin of the offset and further validate the proxy, we investigated the trophic ecology of the asymbiotic scleractinian cold-water coral Balanophyllia elegans, both in a laboratory setting and in its natural habitat. A long-term incubation experiment of B. elegans fed on an isotopically controlled diet yielded a canonical trophic isotope effect of 3.0 ± 0.1 ‰ between coral soft tissue and the Artemia prey. The trophic isotope effect was not detectably influenced by sustained food limitation. A long N turnover of coral soft tissue, expressed as an e-folding time, of 291 ± 15 d in the well-fed incubations indicates that coral skeleton δ15N is not likely to track subannual (e.g., seasonal) variability in diet δ15N. Specimens of B. elegans from the subtidal zone near San Juan Channel (WA, USA) revealed a modest difference of 1.2 ± 0.6 ‰ between soft tissue and skeletal δ15N. The δ15N of the coral soft tissue was 12.0 ± 0.6 ‰, which was ∼6 ‰ higher than that of suspended organic material that was comprised dominantly of phytoplankton – suggesting that phytoplankton is not the primary component of B. elegans' diet. An analysis of size-fractionated net tow material suggests that B. elegans fed predominantly on a size class of zooplankton ≥500 µm, implicating a two-level trophic transfer between phytoplankton material and coral tissue. These results point to a feeding strategy that may result in an influence of the regional food web structure on the cold-water coral δ15N. This factor should be taken into consideration when applying the proxy to paleo-oceanographic studies of ocean N cycling.

Non-mycorrhizal root-associated fungi increase soil C stocks and stability via diverse mechanisms

Abstract. While various root-associated fungi could facilitate soil carbon (C) storage and therefore aid climate change mitigation, so far research in this area has largely focused on mycorrhizal fungi, and potential impacts and mechanisms for other fungi are largely unknown. Here, with the aim of identifying novel organisms that could be introduced to crop plants to promote C sequestration, we assessed the soil C storage potential of 12 root-associated, non-mycorrhizal fungal isolates (spanning nine genera and selected from a wide pool based on traits potentially linked to soil C accrual) and investigated fungal, plant and microbial mediators. We grew wheat plants inoculated with individual isolates in chambers allowing continuous 13C labelling. After harvest, we quantified C storage potential by measuring pools of different origin (plant vs. soil) and different stability with long-term soil incubations and size/density fractionation. We assessed plant and microbial community responses as well as fungal physiological and morphological traits in a parallel in vitro study. While inoculation with 3 of the 12 isolates resulted in significant total soil C increases, soil C stability improved under inoculation with most isolates – as a result of increases in resistant C pools and decreases in labile pools and respired C. Further, these increases in soil C stability were positively associated with various fungal traits and plant growth responses, including greater fungal hyphal density and plant biomass, indicating multiple direct and indirect mechanisms for fungal impacts on soil C storage. We found more evidence for metabolic inhibition of microbial decomposition than for physical limitation under the fungal treatments. Our study provides the first direct experimental evidence in plant–soil systems that inoculation with specific non-mycorrhizal fungal strains can improve soil C storage, primarily by stabilising existing C. By identifying specific fungi and traits that hold promise for enhancing soil C storage, our study highlights the potential of non-mycorrhizal fungi in C sequestration and the need to study the mechanisms underpinning it.

Rapid high-throughput processing of tissue samples for microbiological diagnosis of periprosthetic joint infections using bead-beating homogenization.

Enrichment of periprosthetic tissue samples in blood culture bottles (BCBs) for microbiological diagnosis of periprosthetic joint infections (PJI) is more reliable than the use of an enrichment broth. Nevertheless, the extremely time-consuming homogenization of the samples for BCB processing has so far limited its use, especially in high-throughput settings. We aimed to establish a highly scalable homogenization process of tissue samples for long-term incubation in BCBs. A protocol for homogenization of tissue samples using bead beating was established and validated. In a second step, the use of the homogenate for enrichment in BCBs was compared to the use of thioglycolate broth (TB) in terms of diagnostic accuracy using clinical tissue samples from 150 patients with suspected PJI. Among 150 analyzed samples, 35 samples met the microbiological criteria for PJI. Using BCB, 32 of 35 (91.4%) PJI were detected compared to 30 of 35 (85.7%) by TB. The use of BCB had a lower secondary contamination rate (2/115; 1.7% vs 4/115; 3.5%) but the trend was not significant due to low numbers of samples (P = 0.39). The time to process a batch of 12 samples using the established homogenization method was 23 ± 5 min (n = 10 batches). We established and validated a homogenization workflow that achieves the highest sensitivity in the microbiological diagnostic of PJI. The enrichment of the tissue homogenate in BCBs showed equally good results as the use of enrichment broth and allows semi-automated high-throughput processing while demonstrating lower contamination rates in our study.

Investigation of Potential Effects of Ibuprofen on the Storage Cells and Anhydrobiosis Capacity of the Tardigrade Paramacrobiotus experimentalis

The surge in pharmaceutical consumption, particularly non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, has raised concerns about their presence in aquatic ecosystems. This study investigated the potential ecological impact of ibuprofen, focusing on the ultrastructure of storage cells in the tardigrade Paramacrobiotus experimentalis, renowned for its resilience to environmental stressors. Individuals were exposed to three ibuprofen concentrations (0.1 μg/L, 16.8 μg/L, and 1 mg/L) over 7 and 28 days. Storage cells were examined using light microscopy, transmission electron microscopy, and confocal microscopy. This study also explored ibuprofen’s impact on the process of anhydrobiosis. In the short-term experiment, no ultrastructural changes in tardigrade storage cells were observed across ibuprofen concentrations. However, in the long-term incubation, autophagic structures in storage cell cytoplasm were identified, indicating potential adaptive responses. Individual mitochondria exhibited degeneration, and the rough endoplasmic reticulum displayed slight swelling. No evidence of increased oxidative stress or nuclear DNA fragmentation was observed in any research group. This study elucidates the complex responses of tardigrade storage cells to ibuprofen exposure. The findings emphasize the importance of understanding pharmaceutical impacts on aquatic organisms, highlighting the resilience of tardigrades to specific environmental stressors.

Study in a long-term laboratory experiment of the potential susceptibility to mineralization of organic matter in post-agrogenic light gray soils

Relevance. Since 2021, work has begun in Russia to assess the fertility of unused arable land and its involvement in agricultural circulation. Changes in the farming system of post-agrogenic soils can lead to uncontrolled mineralization of newly formed soil organic matter (SOM) under fallow vegetation and significant CO2 emissions into the atmosphere. Studies of the nature of SOM accumulation under fallow vegetation and assessment of its potential susceptibility to mineralization are relevant, since they can become the basis for the development of agrotechnical methods for returning unused lands to arable circulation with the maximum preservation of their fertility.Methods. In a long-term laboratory incubation experiment, we studied the dynamics of changes in the intensity of basal (BR) and substrate-induced respiration (SIR) in postagrogenic soils to assess the potential susceptibility to SOM mineralization with a change in land use. Layered samples (0–10 and 10–20 cm) were used from the old arable horizons of two fallow plots with different humus conditions. The results of the incubation experiment were compared with the results of assessing the quantitative content and qualitative composition of SOM.Results. In the 0–10 cm layer, the respiration rates are higher than in the 10–20 cm layer. The results of the assessment of the respiration intensity are consistent with the assessment of the qualitative composition of SOM. The accumulation of SOM occurs mainly in the upper part of postagrogenic soils due to mobile easily oxidized organic compounds of a fulvic nature. When developing agrotechnical methods for returning fallow lands to arable circulation, it is necessary to focus primarily on basic processing technologies that ensure maximum preservation of potentially easily mineralized material accumulated in the upper layer.

Utilization of phosphoric acid-modified biochar to reduce vanadium leaching potential and bioavailability in soil

Remediating vanadium (V) polluted soil has garnered widespread attention over the past decade. Yet, few research projects have investigated the stabilization of soil V using modified biochar, so the effects and interacting mechanisms between soil properties and modified biochar for V immobilization and stabilization remain unclear. Hence, this gap is addressed by determining the leaching behavior and mechanisms of soil V on different dosages of phosphoric acid (H3PO4) impregnated biochar (MLBC, 0.5%–4%). The applicability and durability in soil V immobilization was investigated under acid precipitation. The MLBC effect on V bioavailability and mobility was assessed first by CaCl2, Toxicity Characteristic Leaching Procedure (TCLP) and Synthetic Precipitation Leaching Procedure (SPLP) extractions in different periods. The V concentrations significantly reduced in CaCl2, TCLP, and SPLP extract with MLBC at each dosage (30 d), while slight to significant increase in SPLP and TCLP extract V was recorded in a long-term incubation (90 d). Column leaching test further demonstrated the high durability of 4% MLBC in V stabilization under continuous acid exposure. Compared to the control (no-biochar), the accumulated V content in the leaching solution significantly decreased in MLBC-amended soil. Acid soluble fraction of V showed significant negative correlation with both soil organic matter (SOM) and available P, which was positively correlated with pH, suggested that pH, available P and SOM were key factors affecting the bioavailability of V in soil. Moreover, combining with the characterization results of MLBC and amended soil, the results revealed that H3PO4 modified biochar played a vital role on V immobilization and soil improvement by forming electrostatic adsorption, ion exchange, redox reaction or complexation with the increase of functional groups. These revealed an efficient and steady development of soil quality and treatment for soil V contamination, under MLBC operation to soil polluted with exogenous V.

Projected soil carbon loss with warming in constrained Earth system models

The soil carbon-climate feedback is currently the least constrained component of global warming projections, and the major source of uncertainties stems from a poor understanding of soil carbon turnover processes. Here, we assemble data from long-term temperature-controlled soil incubation studies to show that the arctic and boreal region has the shortest intrinsic soil carbon turnover time while tropical forests have the longest one, and current Earth system models overestimate intrinsic turnover time by 30 percent across active, slow and passive carbon pools. Our constraint suggests that the global soils will switch from carbon sink to source, with a loss of 0.22–0.53 petagrams of carbon per year until the end of this century from strong mitigation to worst emission scenarios, suggesting that global soils will provide a strong positive carbon feedback on warming. Such a reversal of global soil carbon balance would lead to a reduction of 66% and 15% in the current estimated remaining carbon budget for limiting global warming well below 1.5 °C and 2 °C, respectively, rendering climate mitigation much more difficult.

Quercetin is a foe in the heart by targeting the hERG potassium channel.

Quercetin is a plant flavonoid known for its pharmacological activities, such as antioxidant, anti-inflammatory, and anti-cancer properties. However, there is limited information available regarding its potential toxicities. A previous study showed that quercetin can inhibit human ether-a-go-related gene (hERG, also named KCNH2) currents, which may lead to long QT syndrome, torsade de pointes (TdP), and even sudden cardiac death. This study aimed to investigate the effects of quercetin on hERG and its potential mechanism. hERG currents and action potential duration (APD) were assessed using the patch clamp technique. Molecular docking was employed to elucidate the binding sites between quercetin and hERG. Transfection of wild-type or mutant plasmids was used to verify the results of molecular docking. Western blot was performed to determine the expression levels of hERG, transcription factor SP1, molecular chaperones HSP70 and HSP90, phosphorylated E3 ubiquitin ligase p-Nedd4-2, serum- and glucocorticoid-inducible kinase (SGK1), and phosphatidylinositol 3-kinase (PI3K). Immunoprecipitation was conducted to evaluate hERG ubiquitination. Quercetin acutely blocked hERG current by binding to F656 amino acid residue, subsequently accelerating channel inactivation. Long-term incubation of quercetin accelerates Nedd4-2-mediated ubiquitination degradation of hERG channels by inhibiting the PI3K/SGK1 signaling pathway. Moreover, the APD of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) is significantly prolonged by 30 μM quercetin. Quercetin has a potential risk of proarrhythmia, which provided useful information for the usage and development of quercetin as a medication.

Effect of cadmium ions on photosystem II oxygen evolution

The paper presents the results of the study of the effect of cadmium (CdSO4) ions on photosystem II (PSII) photochemical activity. The presence of Cd2+ ions in the medium was found to significantly inhibit the oxygen evolution in the thylakoid membranes, indicating a clear correlation between the concentration of Cd2+ ions and the inhibition of PSII photochemical activity. Additionally, the study identified that this inhibition is strongly dependent on the pH level of the medium. The inhibition of PSII photochemical activity occurred quickly at low cadmium concentrations (1.0-30 mM). The study also observed that the inhibition of O2 evolution by Cd2+ ions was time-dependent, with a rapid 20-40% inhibition of oxygen evolution occurring when adding Cd2+. The photochemical activity of PSII was inhibited slowly during long-term incubation. The findings imply that PSII contains at least one high-affinity cadmium binding site. Keywords: Cadmium, photosynthesis, photosystem II, oxygen evolution

Survival of surface bacteriophages and their hosts in in situ deep-sea environments.

The survival of the sinking prokaryotes and viruses in the deep-sea environment is crucial for deep-sea ecosystems and biogeochemical cycles. Through an in situ deep-sea long-term incubation device, our results showed that viral particles and infectivity had still not decayed completely after in situ incubation for 1 year. This suggests that, via infection and lysis, surface viruses with long-term infectious activity in situ deep-sea environments may influence deep-sea microbial populations in terms of activity, function, diversity, and community structure and ultimately affect deep-sea biogeochemical cycles, highlighting the need for additional research in this area.

Impact of Temperature and Moisture on the Decomposition of Peat-Forming Plants: Results of a Two-Year Incubation Experiment

The decomposition rate of plant residues is determined by both abiotic (temperature, moisture) and biotic factors (biochemical composition). To separate the contribution of each factor to the decomposition process, long-term incubation experiments under controlled conditions are required. Two-year incubation experiments were conducted with various types of peat-forming plants (Sphagnum fuscum, Chamaedaphne calyculata, Eriophorum vaginatum, and a mixed sample consisting of 60% Sphagnum fuscum and 40% Chamaedaphne calyculata). The experiments were carried out at temperatures of 2, 12, and 22 °C, with varying moisture levels (W = 30, 60, and 90% of their water-holding capacity). In all plant samples, the highest rates of C(CO2) emission (DecR) were observed in the initial stages of decomposition. The cumulative carbon loss (Ccum) during the experiment ranged from 45 to 196 mgC/g of plant material at 22 °C and 23 to 156 mgC/g of plant material at 2 °C. The decay constant (k) for all plant samples increased with rising temperature. The results of the three-way ANOVA showed that the influence of the examined factors on the cumulative losses of C(CO2) decreased in the following order: the type of plant > temperature > moisture. Throughout the experiment, the influence of the type of plant and moisture on DecR increased, while the effect of temperature decreased. The highest temperature sensitivity (Q10 = 0.71–6.19) was observed in the low-temperature range (2–12 °C) during months 4 to 6 of incubation. These results are relevant for modeling and predicting the rate of transformation of peat organic matter under changing climatic conditions.

Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase.

Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

A Novel Milli-fluidic Liver Tissue Chip with Continuous Recirculation for Predictive Pharmacokinetics Applications

A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low- and high-clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)–based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low- and high-clearance compounds for at least 12 days, and clearance was estimated for a range of compounds with high in vitro-in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.Graphical

Viruses Demonstrate Selective Survival During Simulated Anaerobic Digestion of Plant Biomass

Objective. This research aimed at laboratory simulation of anaerobic digestion of plant biomass contaminated by a mixture of viruses with a wide host range to evaluate if viruses can retain their infectivity and pose a biohazard during the use of plant-based reusable resources such as digestate. Methods. Anaerobic digestion was simulated in air-tight desiccators using anaerobic bags and indicator strips to ensure proper conditions, and using fresh biomass of naturallyvirus-infected plants. In one of the variants, a commercial mixture of anaerobic bacteria and enzymes was also added to see if it would be more efficient in terms of plant digestion and virus inactivation. For serological detection of the tomato mosaic virus, cucumber mosaic virus, and potato virus Y, DAS-ELISA was used with commercial diagnostic kits and positive controls. Absorbance values were measured at 405 nm. Total RNA was extracted using a RNeasy Plant Mini kit following the manufacturer’s instructions. Molecular detection of viruses was carried out by two-step RT-PCR using specific oligonucleotide primers available from the literature. To check if the viruses can retain their infectivity after the anaerobic digestion of plant biomass waste, a bioassay was used where Nicotiana rustica plants producing necrotic lesions upon infection with many viruses were mechanically inoculated at the stage of 2 true leaves. For statistical processing of data, the mean and standard error were calculated using Microsoft Excel software. Results. After prolonged anaerobic digestion, tomato mosaic virus was still detected in plant residues, but it is important to note that it was also found in high concentration in the liquid phase. Hence, during the anaerobic digestion, a significant proportion of the virus was successfully eluted from plant residues into the digestate. Interestingly and contrary to tomato mosaic virus, cucumber mosaic virus has not practically eluted to the liquid phase. On the other hand, potato virus Y was supposedly completely decomposed during the anaerobic digestion of plant biomass waste, as it was not detected either by ELISA or by PCR. It is likely that not all of the investigated viruses represent a potential biohazard in the context of the use of renewable resources. RT-PCR outcomes showed the presence of tomato mosaic virus and cucumber mosaic virus both in the solid and the liquid phases, augmenting the ELISA data. Lastly, bioassay results have confirmed that at least tomato mosaic tobamovirus may escape the aggressive process of waste decomposition and remain able to infect host plants raising issues about the safety of reusable plant resources. Conclusions. The survival of plant viruses during plant biomass waste processing was studied in the context of biological safety. The preservation of serological markers and genetic signatures of model viruses in the process of anaerobic digestion has been confirmed. For the first time, it was proven that tomato mosaic virus may preserve its infectivity during long-term incubation of plant biomass waste in anaerobic conditions, which requires further attention when using renewable resources. On the example of PVY, it has been shown that potyviruses do not survive during anaerobic digestion and therefore may not pose an epidemic risk in using processed renewable resources. It has been demonstrated that artificial introduction of anaerobic microorganisms does not have a decisive effect on the stability of the studied model viruses.

Dissolved organic matter and nutrient processing in organic-rich subterranean estuaries: Implications for future land use and climate scenarios

The subterranean estuary (STE) is a recognized reactive biogeochemical zone at the groundwater–seawater interface in coastal regions. However, few studies have explored dissolved organic matter (DOM) and nutrient processes in buried organic-rich STEs (with up to 20 % of organic matter content), despite their potential contributions to coastal water hypoxia and greenhouse gas emissions. This study conducted controlled laboratory experiments to investigate the physicochemical controls on DOM and nutrient processing in organic-rich STEs, considering variations in groundwater nitrate (NO3–) concentrations, salt concentration, seawater ions, and incubation times. Results showed elevated levels of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) in pore water, primarily from particulate organic matter (POM) degradation that originated from decayed vascular plants. POM-to-DOM degradations further resulted in the accumulation of ammonium (NH4+) and highly unsaturated compounds in all treatments. The addition of high-ionic-strength treatments (e.g., seawater and sodium chloride [NaCl] solution) reduced DOC solubility and selectively removed dissolved aromatic compounds in porewater. The cation-rich seawater shifted the porewater NH4+ generation pathway from predominantly biological (re)mineralization in freshwater to abiotic (desorption via ion exchange) pathways in the saline water setup. Orthophosphate (PO43−) dynamics were likely to be influenced by the iron-rich sediment surface in both freshwater and saline water scenarios. Our experiments further indicated that the long-term incubation enriched DOM concentration with a high degradation state. This work demonstrated that organic-rich STEs are significant sources of less labile DOM and NH4+ to coastal waters. In the context of global climate change, our results illustrate the sensitivity of DOM and nutrient biogeochemical cycling in STEs to salinization and altered residence time, consequently affecting the quantity and quality of DOM and nutrients transported to coastal waters.